The Renal Differentiation and Neoplasia Section studies inductive signaling in tissue development/morphogenesis and, in parallel, its dysregulation in tumorigenesis with emphasis on the ligands that mediate normal tissue interactions and the pathways and targets that are activated in response to signaling. Our focus has been on development of the urogenital tract, which features reciprocal interactions between two distinct mesodermal progenitors, highly coordinated tissue movements, mesenchymal-epithelial transition (MET), integration of structures from different lineages, reiterative cycles of development, and a tumor that caricatures nephrogenesis. More specifically we are interested in the signaling mechanisms that direct metanephric mesenchyme (MM) to convert to the epithelia of the nephron. Wilms tumor (WT) is characterized by an expanded blastemal/progenitor population with a restricted capacity for epithelial conversion (MET). It has been our long-term goal to identify targets on which WT cells depend for survival or dysregulated signaling that can be reprogrammed to allow tumor cells to differentiate to a more benign phenotype. With my retirement, efforts have been focused on bringing current studies to a reasonable and rapid conclusion leading to publication of the results. Accordingly, we have continued an assessment of cell conditions which allow for the robust expansion of cultured Wilms tumor cells for real-time testing of personalized options for therapeutic intervention. We previously defined conditions for the propagation of nephronic progenitors and applied similar conditions to studies of Wilms tumor. With minor modifications we have successfully grown Wilms tumor cells from primary tumors removed from multiple patients. These cells retain the expression of several stemness and WT-associated genes, such as, SIX2, ALDH1, and PAX2. They are stable for several passages and retain a tumorigenic phenotype based upon their ability for anchorage-independent growth. Cultured cells from these tumors have now been xenografted into NSG mice to further assess tumorigenic behavior, and one of the WT cultured cell preparations has formed tumors in mice. These tumors can be continuously passaged and retain their embryonal WT character histologically. All 5 cultured WTs readily form organoids when placed in nonadherent culture wells, but these conditions proved inferior to monolayer cultures in sustaining the expression of stemness markers with passage. Given the belief that these cells provide the driving force for WTs, the massive expansion of this population in culture witnessed in these studies may permit the development of personalized therapeutic screening for individual WT patients. This is a major advance in the field, as heretofore, failed efforts to propagate WT cells in culture have led some to speculate that they cannot be sustained except in xenografts. I am also in the process of revising a manuscript which describes an aberrant phenotype in the urinary tract associated with Wnt5a ablation, i.e., hydronephrosis. In these studies we show that hydronephrosis occurs as the result of a blockage in urine flow at the time renal function becomes active, causing apoptosis of the medullary region in the kidney. The blockage occurs at the interface between the ureter and bladder when cells in the common nephric duct fail to apoptose. Gene expression analysis revealed that Shh was increased in the pericloacal region in Wnt5a mutants, and we could rescue the phenotype with Shh haploinsufficiency. We also determined that constitutive activation of HH signaling yielded hydronephrosis and that Shh, which is produced by the cloacal epithelium, signals directly to the common nephric duct to sustain the tubular structure, preventing its degeneration to allow for ureter insertion into the bladder. This work demonstrates for the first time that Wnt5a suppresses Shh levels during development and possibly also during tumorigenesis, since Wnt5a expression is inversely regulated in tumorigenesis in such tissues as kidney and female reproductive tract. Taking this work one step further, I have ablated Wnt5a expression from all cells that respond to HH signaling during development (Gli1-Cre-ERT2). With the incorporation of a floxed allele for LacZ, I can identify those populations which are responding to Gli activation. It appears from these studies that Wnt5a is a global regulator of HH signaling throughout the embryo. Finally, I am also engaged in ongoing collaborative studies on Fgf8 with Mark Lewandoski's lab. These involve efforts to better understand its function in nephronic progenitor maintenance. This has been pursued using mouse genetics and cell culture models. To this end I have successfully demonstrated the partial rescue of progenitor survival in Fgf8 loss-of-function mutant mouse embryos lacking pro-apoptotic factors bax and bak. In another collaboration, we are evaluating the role of FgfrL1 in Fgf8 signaling in the developing kidney, since this receptor has greatest affinity for Fgf8 yet lacks a cytoplasmic signaling domain. Finally we seek an explanation for the absence of a cortical phenotype in Fgf8 mutant kidneys, when a kidney-specific Cre is used rather than mesodermal localized TCre. This suggests that Fgf8 signaling to the kidney is mediated at least in part by expression from adjacent tissues rather than the metanephros itself - possibly the paraxial mesoderm.